This invention is in the field of tissue and organ transplantation and immunological tolerance.
The induction of graft specific immunological tolerance is a much sought after goal as it would allow the transplantation of cells or organs without generalized immunosuppression. While a variety of procedures to induce graft specific tolerance have been proposed, non-specific immunosuppressive agents such as cyclosporin A and FK506 remain the agents of choice for treatment of patients undergoing transplantation. These agents allow the transplantation of xe2x80x9cforeignxe2x80x9d organs, but their immunosuppressive effect is accompanied by numerous side effects.including opportunistic infection, increased rate of neoplasm, and liver and kidney toxicity. Moreover, cyclosporin rarely allows long term graft function; the 10 year survival rate for allogeneic heart and kidney transplants has stabilized at about 50% [Kaufman and Ildstad (1994) Therapeutic Immunol. 1:101].
The invention is based on the discovery of six novel porcine MHC class I genes, the elucidation of their nucleotide sequences (SEQ ID NOS:1-6), and the deduction of the amino acid sequences (SEQ ID NOS:7-12) that they encode. These genes demonstrate a high degree of homology between each other and to MHC class I genes of other mammalian species. In addition, they are efficiently expressed when transferred to xenogeneic cells. These properties provide the basis for their use in establishing immunological tolerance in prospective transplant recipients prior to grafting with porcine cells, tissues or organs. Furthermore the genes and their products provide the components necessary for a variety of in vitro assays for screening transplant recipients and donors. Moreover, the polypeptides produced by the MHC class I genes of the invention can be used to prepare and screen a variety of useful antibodies.
Specifically, the invention features nucleic acid molecules that encode porcine MHC class I polypeptides which are at least 95% identical to the polypeptides PA1 (SEQ ID NO:7), PC1 (SEQ ID NO:8), PA14 (SEQ ID NO:10), or PC14 (SEQ ID NO:11). Also included in the invention are nucleic acid molecules that encode the porcine MHC class I polypeptides PA1 (SEQ ID NO:7), PC1 (SEQ ID NO:8), PD1 (SEQ ID NO:9), PA14 (SEQ ID NO:10), PC14 (SEQ ID NO:11), or PD14 (SEQ ID NO:12). The latter nucleic acid molecules can be pa1 (SEQ ID NO:1), pc1 (SEQ ID NO:2), pd1 (SEQ ID NO:3), pa14 (SEQ ID NO:4), pc14 (SEQ ID NO:5) or pd14 (SEQ ID NO:6).
The invention encompasses expression vectors containing these nucleic acid molecules, cell lines transfected with the expression vectors, the polypeptides encoded by the above nucleic acid molecules and antibodies specific for the polypeptides.
The invention features variant porcine MHC class I polypeptides in which one ore more of xcex11, xcex12, or xcex13 is deleted or replaced by the corresponding domain of a human or murine MHC class I polypeptide and variant porcine MHC class I polypeptides in which one or more amino acid residues has been replaced by the corresponding amino acid residue of a human MHC class I polypeptide. The invention also features the nucleic acid molecules encoding these variant MHC class I polypeptides.
Also provided is a method of preventing porcine graft rejection in a patient in which antibodies directed against a porcine MHC class I polypeptide are administered to a transplant recipient prior to or in conjunction with transplantation of porcine tissue or cells. This treatment may be given with or without immunosuppression. In addition, a method for inducing specific immunological tolerance in prospective transplant recipients is included in the invention. This method involves administering to the patient autologous cells transfected with and expressing the porcine MHC class I genes of the invention. These autologous cells are those expressing neither MHC class II polypeptides nor costimulatory molecules such as B7, e.g., fibroblasts, myoblasts, or keratinocytes. This prophylactic treatment may also be given with and without immunosuppression and may be used in prospective recipients of porcine cells, for example, pancreatic islets, hepatocytes, cardiac cells, corneal cells, neural cells, retinal cells, or myoblasts or porcine organs.
Also featured in the invention are assays to monitor tolerance in transplant recipients and for selecting an appropriate pig as a tissue donor. In one aspect, these assays involve testing patients for antibodies to porcine MHC class I polypeptides, including those of the invention. In a second aspect, lymphoid cells from the patient are tested for the presence of cytotoxic T lymphocytes by culturing the lymphoid cells with cells expressing the porcine MHC class I polypeptides or with purified porcine MHC class I polypeptides. The cultured cells are then tested for specific cytolytic activity against target cells expressing the porcine MHC class I polypeptide of interest. Also with the invention are methods for identifying, in a patient after tolerization, T cells (e.g., CD4+ cells) which react with porcine MHC class I. These methods entail contacting the T cells with a porcine MHC class I polypeptide and determining whether the T cells proliferate.
As used herein, xe2x80x9cpa1, pc1, and pd1xe2x80x9d are nucleic acid molecules corresponding to the aa, cc and dd alleles, respectively, of a porcine MHC class I gene referred to herein as p1.
As used herein, xe2x80x9cpa14, pc14, and pd14xe2x80x9d are nucleic acid molecules corresponding to the aa, cc and dd alleles, respectively, of a porcine MHC class I gene referred to herein as p14.
As used herein, xe2x80x9cPA1, PC1, PD1, PA14, PC14, and PD14xe2x80x9d are porcine MHC class I polypeptides encoded by pa1, pc1, pd1, pa14, pc14, and pd14, respectively.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, will control. In addition, the materials, methods, and examples described herein are illustrative only and not intended to be limiting.
Other features and advantages of the invention, e.g., prevention of graft rejection, will be apparent from the following detailed description, from the drawings and from the claims.